The present invention concerns functional layers of high precision and their production, the use of N-acyl-N-alkyl-glycinates, N-acyl-taurates and/or N-acyl-glutamates to produce these functional layers as well as test strips and in particular diagnostic test strips which carry at least one functional layer of high precision according to the invention in their test elements.
So-called carrier-bound tests are often used for the qualitative or quantitative analytical determination of components of body fluids in particular of blood. In these the reagents are present on or in corresponding layers of a solid test carrier which is contacted with the sample. The reaction of liquid sample and reagents leads to a detectable signal especially to a change in colour which can be evaluated visually or with the aid of an instrument, usually by reflection photometry.
Test carriers are frequently in the form of test strips which are essentially composed of an oblong support layer made of plastic material and detection layers mounted thereon as test fields. However, test carriers are also known which are designed as small quadratic or rectangular plates. In the following description the term xe2x80x9ctest stripsxe2x80x9d is also intended to encompass test carriers which do not have a strip shape.
Test carriers of the above-mentioned type are for example known from the German Patent document 21 18 455. This describes diagnostic test carriers for the detection of analytes in liquids which are composed of a support layer and at least one detection layer containing the detection reagents. That surface of the detection layer that does not adjoin the support layer is provided with a cover layer. The cover layer can be composed of a fine-meshed network in the form of a fabric, knitted fabric or fleece. Plastic fabrics are stated as preferred networks in order to achieve a rapid wetting of the detection layer with sample liquid and to avoid interfering chromatographic effects. In order to detect an analyte in a liquid, such a diagnostic test carrier is dipped into a corresponding liquid, preferably urine. In this manner the detection layer comes into contact with a very large excess of liquid which cannot be taken up by the test carrier. However, depending on the duration of contact of the detection layer with the liquid to be examined different colour intensities can be observed.
As a rule the results obtained are more positive the longer the contact time is. Therefore a correct quantitative analyte determination is not possible in this manner when there is a large excess of sample.
On the other hand a sample volume that is too small for a test carrier construction is a frequent cause for false measured values in diabetes monitoring i.e. the regular control of the blood of diabetics for the content of glucose.
Test carriers which require as little volume as possible are therefore the goal of diverse current developments. However, such test carriers do not only have to yield correct measured values with very small sample volumes of about 3 xcexcl, but they must also reliably operate with relatively large sample volumes of about 15-20 xcexcl and must hold the sample liquid. Hygienic problems could occur if the liquid runs out of the test carrier for example if potentially infectious foreign blood is measured or if the test carrier is measured with an apparatus when there is a risk of contaminating the measuring instrument.
Test strips are known from DE-A-3042857 which have a sample distribution layer (spreading layer) on their analytical elements which has the function of uniformly distributing sample liquid applied as a spot over the entire test element. This spreading layer is composed of a cloth or a foam layer which is hydrophilized by impregnation with a wetting agent and is either pressed onto the upper gelatin layer of the analytical element which is still wet or is attached thereto by means of an additional adhesive layer.
The analytical elements of these known test strips which are referred to synonymously in the literature and in the following description as test elements, test fields, detection elements, detection fields or as detection layers have two or more layers which contain the reagents necessary to detect and quantitatively determine the analyte (in this case urea) or auxiliary substances such as radiation-absorbing substances. Such layers are referred to as functional layers in the following.
Diagnostic test carriers in the form of test strips which offer a considerable advance with regard to reproducibility of the test results even when different sample volumes are applied and with regard to hygienic handling are known from EP-A-0 821 233.
They contain a support layer with a detection layer arranged thereon having one or several functional layers which contain reagents required to determine the analyte in a liquid sample and a hydrophilic, but not capillary-active, relatively coarse-meshed overlay made of a network covering the detection layer which is larger than the detection layer and is attached to the support layer on both sides of the detection layer but in contrast rests directly on the detection layer without attachment i.e. essentially is in contact with the whole surface of this without a gap.
This network rapidly passes sample liquid applied to its surface onto the underlying detection layer and leads, with the aid of a foil layer that covers the boundary regions of the network, a sample excess which may be present into the boundary regions of the network which extend beyond the detection layer. In this manner small amounts of sample are made completely available to the detection layer but false-positive results are avoided.
Developments in the prior art apparently have the aim of achieving quantitative determinations of the analytes of interest that are as accurate as possible using test strips with smaller and smaller sample volumes. The improvement of the test strip construction also goes hand in hand with a reduction of the detection areas used for the analysis. Thus for example in a known instrument that is frequently used for the routine determination of blood sugar, the GLUCOTREND(copyright) instrument, only an area of ca. 1 mm diameter of the detection layer is evaluated.
An advantage of this trend is that small amounts of sample result in readily detectable colour signals on the small areas, but there is a risk that even slight local differences in the structure of the functional or detection layers can lead to serious measuring errors.
Conventional functional layers can contain a skeleton of a fibrous or non-fibrous porous material which incorporates the reagents required to detect the analytes and auxiliary substances and additives.
It is basically necessary to use those materials for the functional layers which are able to take up the liquid to be examined together with the components contained therein. These are so-called absorbent materials such as fleeces, fabrics, knitted fabrics, membranes or other porous plastic materials which can be used as a skeleton for the layer and of course decisively determine the structure and dimensions of the layer. The materials that come into consideration for the detection layer must of course also be able to carry reagents which are required to detect the analyte to be determined. In the simplest case all reagents required to detect the analyte are located on or in such a skeleton material.
Skeleton materials that are frequently used for the functional layer are papers, the above-mentioned textile fabrics made of natural or synthetic fibres or porous plastic materials such as membranes and in particular asymmetric porous membranes in which case the sample liquid to be examined is usually applied to the large-pored side of the membrane and the analyte is determined on the fine-pored side of the membrane. Particularly preferred porous membrane materials are polyamide, polyvinylidene difluoride, polyethersulfone or polysulfone membranes, in particular polyamide 66 membranes and hydrophilized asymmetric polysulfone membranes are used. The reagents for the determination of the analyte to be detected are usually incorporated into the aforementioned materials by impregnation or are applied to one side by coating. When coating asymmetric membranes it is preferable to coat the fine-pored side.
By nature the skeleton materials introduce an inhomogeneity in the layers which is averaged out when the colour signals generated by the analytes are evaluated over a large area but can interfere as the areas that are evaluated become smaller.
Hence various approaches have been made to generate skeleton-free functional layers of high precision i.e. with the fewest and smallest possible local inhomogeneities.
In this connection it is important that the layers flow well and adhere well to the bases. The aim is that the surface tension of the layer materials should be reduced or should approximate each other. For this the addition of organic solvents such as 1-hexanol or of wetting agents has been proposed. Another approach is to treat the surface of the respective base on which the layer is to be applied by plasma discharge (corona treatment). A combination of these measures is also possible.
However, these measures of the prior art have considerable disadvantages. Organic solvents such as 1-hexanol cannot be used in high concentrations since they are poorly soluble in water which is the preferred liquid phase for coating compounds. Water-miscible solvents such as acetone also lower the surface tension of the coating compounds but can only be used to a limited extent since they solubilize the plastic foils used as a base for the first layer which bends them and also bends the functional layers that are obtained. Such layers cannot be correctly processed and measured.
Wetting agents cannot be generally used and there is a special limitation when producing glucose detection layers since they must neither denature the enzyme glucose-dye-oxidoreductase (Gluc-DOR) that is used nor should they lyse the erythrocytes in the applied blood sample since otherwise the red colour of the measuring side of the detection layer of the test strips would considerably hinder the detectability and measurability of the colour signal of the detection reaction.
Corona treatments of the bases are primarily an additional process step and moreover require a corresponding expensive apparatus which represents a trouble-prone part of the production line.
There is thus still an urgent need to be able to produce skeleton-free functional layers of high precision i.e. those which have no or the fewest and smallest possible local irregularities.
The present invention addresses this requirement.
This invention concerns a skeleton-free functional layer of high precision comprising a film composed of a natural or synthetic film-forming polymer (film former), one or several compounds enabling the function of the layer, optionally auxiliary substances and/or additives which is characterized in that the layer contains wetting agents of formula I, II and/or III
R1xe2x80x94COxe2x80x94N(R2)xe2x80x94CH2xe2x80x94COOMexe2x80x83xe2x80x83(I)
R1xe2x80x94COxe2x80x94N(R2)xe2x80x94CH2xe2x80x94CH2xe2x80x94SO3Mexe2x80x83xe2x80x83(II)
HO2Cxe2x80x94CH2xe2x80x94CH2xe2x80x94CH(NHxe2x80x94COR1)xe2x80x94CO2Mexe2x80x83xe2x80x83(III)
in which R1 is a preferably straight-chained or slightly branched aliphatic residue with 9 to 23 C atoms, in particular with 11 to 19 C atoms which is saturated or has one to three double bonds,
R2 is a preferably straight-chained or a slightly branched alkyl residue with 1 to 8, preferably 1 to 4 C atoms and
Me represents hydrogen or a metal atom.
R1 is preferably the aliphatic chain of lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid or isomers thereof and
R2 is methyl or ethyl.
Compounds of formulae I, II and III are of particular economic importance and of very good efficacy in which R1 represents a quantity of alkyl residues in which the structure and proportion of the individual alkyl residues in the mixture corresponds to the structure and abundance in natural fats.
Compounds of formulae I, II and III are particularly preferred in which R1xe2x80x94CO is an oleoyl, cocoyl or tallow fatty acid residue and R2 is methyl.
The metal atom representing Me is expediently selected such that the compounds of formulae I, II and III are water-soluble. Me preferably denotes an alkali metal atom and in particular a sodium or potassium atom.
Skeleton-free functional layers according to the invention can contain a mixture of compounds of formulae I, II and/or III as a wetting agent.
For economic reasons skeleton-free functional layers according to the invention are particularly preferred which contain commercial products of formula I or II. Very suitable commercial products are for example sodium N-oleoyl-sarcosinate, which is readily obtainable from N-oleoyl-sarcosine (e.g. (copyright)Crodasinic O from the Croda Company, Nettetal, Germany) and NaOH and sodium N-methyl-N-oleoyl-taurate (e.g. (copyright)Geropon T 77 from the Rhone-Poulenc Chimie Company, Paris, France) and monosodium N-cocoyl-L-glutamate (e.g. (copyright)Aminosoft CS-11 from the Ajinomoto Company, Tokyo, Japan).
It is very surprising that the high precision of the functional layers according to the invention is already achieved when they contain a total of 0.0075 to 2.5% by weight, preferably 0.01 to 2.0% by weight, in particular 0.03 to 1.0% by weight of the wetting agents of formulae I, II and/or III.
The high precision of the functional layers according to the invention results in a considerable increase in the accuracy of qualitative and quantitative determinations of analytes when these are incorporated into test strips as detection layers. Coating compounds according to the invention containing compounds of formulae I, II and/or III also have a considerably lower viscosity than coating compounds of the same composition but without compounds I, II or III and in many cases the extent of the decrease in viscosity is all the larger the higher the viscosity of the coating compound before the addition of compounds I, II or III. This results in a reduction in the differences in viscosity between coating compounds of different compositions so that the viscosity-dependent processing properties of various compositions are equalized which is a major advantage for example for the mechanical pouring of the layers.
It is observed visually that coating compositions according to the invention xe2x96xa1xhibit a mirror-like surface shortly after their application and before drying. Hence they flow and spread very well on the support foil or on the already present first (dry) layer. The surface of layers without addition of compounds I, II or III appears visually to be considerably more uneven.
The functional layers according to the invention are preferably used as detection layers or are a component of detection layers in test strips especially diagnostic test strips and contain as such reagents or auxiliary substances and/or additives for the qualitative detection or quantitative determination of analytes in addition to the film formers.
Details of the composition of the functional layers according to the invention are as follows:
The functional layers are produced from dispersions or emulsions of polymeric film formers. Dispersion film formers contain microscopic polymer particles which are insoluble in the carrier liquid (usually water) which are dispersed in a fine dispersion in the carrier liquid. If the liquid is removed by evaporation during film formation then the particles approach each other and finally touch. The large forces which occur in this process and a reduction of the surface energy associated with the film formation result in a growth of the particles to a largely continuous film layer. Alternatively it is also possible to use an emulsion of the film former in which it is dissolved in a solvent. The dissolved polymer is emulsified in a carrier liquid which is imiscible with the solvent.
Polyvinyl esters, polyvinyl acetates, polyacrylic esters, polymethacrylic acid, polyvinylamides, polyamides and polystyrene are suitable as polymers for such film formers. Mixed polymers e.g. of butadiene, styrene or maleic acid ester are also suitable in addition to homopolymers.
If a functional layer according to the invention is combined with an additional inventive or also non-inventive functional layer to form a detection layer then the functional layers can be produced from coating compositions which contain the same polymeric film former or they can be produced from coating compositions which contain different polymeric film Formers.
However, so-called open films also come into consideration for the functional layer as described for example in EP-B-0 016 387. For this solids in the form of fine insoluble organic or inorganic particles are added to an aqueous dispersion of film forming organic plastics and the reagents required for the detection reaction are additionally added. Suitable film formers are preferably organic plastics such as polyvinyl esters, polyvinyl acetates, polyacrylic esters, poly-methacrylic acid, polyacrylamides, polyamides, poly-styrene, mixed polymers for example of butadiene and styrene or of maleic acid ester and vinyl acetate or other film forming natural and synthetic organic polymers as well as mixtures of the same in the form of aqueous dispersions. The dispersions can be spread on a base to form a uniform layer which results in a water-resistant film after drying. The dry films have a thickness of 10 xcexcm to 500 xcexcm, preferably of 30 to 200 xcexcm. The film can be used together with the base as a support or it can be mounted on another support for the detection reaction. Although the reagents required for the detection reaction are normally added to the dispersion used to produce the open films, it may also be advantageous to impregnate the final film with the reagents after its production. It is also possible to preimpregnate the filling materials with the reagents.
An additional example of a preferred detection layer according to the invention is a film layer as described in WO-A-92 15 879. This layer is produced from a dispersion or emulsion of a polymeric film former which additionally contains a pigment, a swelling agent and the detection reagent in a homogeneous dispersion. Polyvinyl esters, polyvinyl acetates, polyacrylic esters, polymethacrylic acid, polyvinylamides, polyamides and polystyrene are particularly suitable as polymeric film formers. In addition to homopolymers mixed polymers e.g. of butadiene, styrene or maleic acid ester are also suitable. Titanium dioxide is a particularly suitable pigment for the film. The swelling agent used should have particularly good swelling properties and methyl vinyl ether maleic acid copolymer is particularly recommended.
In addition to the film formers and the reagents for the analyte detection, auxiliary substances and/or additives are very important as a component of the functional layers according to the invention. They serve to adapt the layers to special requirements for example to improve the detectability of the colour reaction, stabilize the reagent systems and/or to prepare the sample liquid by a filter action, e.g. by separating certain components which would interfere with the detection. Examples of such additives are pigments with selected refractive indices, swelling agents or non-porous or porous fillers such as kieselgur (diatomaceous earth).
By adding a good swelling agent (i.e. a substance which increases its volume by uptake of water) one not only obtains layers which are relatively rapidly penetrated by sample liquid but which, despite this opening effect of the swelling agent, have good erythrocyte and additionally also blood pigment separation properties. The swelling properties should be so good that for a test for which the rate of colour formationxe2x80x94such as for example a glucose detection reactionxe2x80x94largely depends on the penetration of the sample liquid through the layer, the optically detectable reaction is measurable after a maximum of one minute. Xanthan gum and methyl vinyl ether maleic acid copolymers have proven to be particularly suitable swelling agents.
Reagent systems for the detection of particular analytes by colour formation are known to a person skilled in the art. It is possible that all components of the reagent system are located in one film layer. It is, however, also possible that the components of the reagent system are divided between the two film layers. Advantageously the colour forming reagent system is at least partly located in the first film layer.
Colour formation is understood in the scope of the present invention not only to mean the Transition from white to coloured but also any colour change whereby of course those colour changes are particularly preferred which are associated with the largest possible shift of the maximum absorption wavelength (xcex max).
The layers according to the invention are produced on a support. This is advantageously a preferably transparent foil as a base support or a foil already provided with an inventive or non-inventive layer. Plastic foils come into particular consideration as the base support which are liquid impermeable. Polycarbonate foil has proven to be particularly preferred.
The thickness of the functional layers according to the invention is usually not more than 0.1 mm, preferably not more than 0.05 mm.
In addition to the skeleton-free functional layers of high precision according to the invention that are described above, the use of compounds of formula I and/or II in which the residues R1, R2, R3 and Me have the meanings stated in claim 1 to produce skeleton-free functional layers of high precision is also a subject matter of the present invention.
The invention also concerns a process for the production of skeleton-free functional layers of high precision by coating a support with a liquid or paste-like coating composition composed of a liquid solvent or dispersant, a solution, a dispersion or a redisperseble or soluble preparation of a natural or synthetic film forming polymer (film former), one or several compounds enabling the function of the layer and optionally auxiliary substances and/or additives and subsequently removing the liquid solvent or dispersant, characterized in that the liquid or paste-like mixture contains a wetting agent of formulae I, II and/or III in which the residues R1, R2 and Me have the meanings stated above.
Solvents in the sense of this description of the process are also so-called apparent solutions i.e. systems of solvent and high-molecular substances which have no phase boundary but exhibit a Tyndall effect. The term dispersion is intended to include all systems composed of a continuous liquid phase, the dispersant and a discontinuous phase that is finally dispersed therein i.e. also emulsions. Suitable groups of natural and synthetic film formers have already been described above in connection with the description of the materials of the functional layers according to the invention.
The liquid or paste-like coating composition used in the process according to the invention contains a total of 0.0075 to 2.5% by weight, preferably 0.01 to 2.0% by weight, in particular 0.03 to 1.0% by weight relative to the weight of all components of the coating composition with the exception of water, of wetting agents of formulae I, II and/or III in which the residues R1, R2 and Me have the meanings stated above.
The support on which the layer according to the invention is generated can already carry one or several inventive or non-inventive functional layers.
The coating can be carried out by all known coating methods which enable a metered application of the coating composition in particular by pouring, by spreading using brushes, paint brushes or knife coating or by roller application or by combinations of these methods. Thus a layer applied by roller can be levelled by brushing or targetted streams of air (so-called air-brushing); blades of various known constructions can be used to remove a possible excess of applied coating composition.
If the support material or/and the other components of the coating composition do not impose other constraints, the liquid phase of the coating composition can be expediently removed at temperatures between room temperature and the boiling point of the liquid phase, preferably at temperatures of about 40 to 80xc2x0 C.
The present invention additionally concerns test strips composed of axe2x80x94as a rule flexiblexe2x80x94flat-shaped carrier on which one or several test elements are arranged next to one another in a test region which each comprises one or several functional layers which rest on top of one another and optionally are covered by an overlay made of a spreading material which are characterized in that at least one of the functional layers is a skeleton-free functional layer of high precision according to the invention.
The test elements composed of one or several functional layers can, as already mentioned above, also be referred to as test fields, detection elements, detection fields or as detection layers.
These terms are also used synonymously in the following description.
With regard to work economy those test strips according to the invention which have two single-layer or multilayer test fields for the same or different analytes which directly adjoin one another or are separated by a gap are particularly advantageous.
If, as is preferred, the test strips are used for diagnostic purposes, then the test fields contain reagents in their functional layers for the detection of a diagnostically utilizable analyte.
The flat-shaped carrier (support layer) of the test strip according to the invention is advantageously composed of a material which does not take up the liquid to be examined. These are so-called non-absorbent materials of which plastic foils for example made of polystyrene, polyvinyl chloride, polyester, polycarbonate or polyamide are particularly preferred. However, it is also possible to impregnate absorbent materials such as wood, paper or cardboard with water-repellent agents or to coat them with a water-resistant film where silicone or hardened fats can be used as hydrophobizing agents and for example nitrocellulose or cellulose acetate can be used as film formers. Metal foils or glass are suitable as additional support materials.
Since the visual assessment and/or the apparative measurement of the test results which are usually in the form of colour changes of the detection layer usually takes place from the side of the detection element opposite to the sample application site, it is necessary that the test strip support is composed of a transparent material and/or has a hole in the region of the test field which is covered by the detection layer. The detection layer but at least the reaction zones of the detection layer are then visible through the perforation. However, several reaction zones of the detection layer may also be visible through one hole.
Especially in preferred test strips with two or several detection elements arranged next to one another, the perforation of a diagnostic test carrier according to the invention can also be composed of two or several holes of the same or different shape which can be used to determine analyte (one or several analytes). Various detection layers or only one detection layer with several reaction zones can be arranged above the holes so that through each hole one detection layer or one reaction zone can be observed.
In a preferred embodiment of the diagnostic test carrier according to the invention there is a hole in the support layer below a detection layer through which the detection layer or a reaction zone can be observed which has a somewhat smaller diameter than the smallest linear extension of the detection layer so that the detection layer rests on the support layer outside the hole and can be attached to the support layer by means of a thin adhesive tape. The detection layer is usually adequately attached by double-sided adhesive tapes arranged on both sides and by the spreading layer lying over the detection layer and its attachment to the support layer.
In order to be able to measure the test reaction simply but nevertheless reproducibly, the test strip support advantageously has adjustment marks e.g. notches or holes which engage in corresponding adjusting elements of the measuring instrument used e.g. a (copyright)GLUCOTREND instrument and thus ensure a correct position of the test strip in the measuring instrument.